Despite the recent success of clinical organ transplantation, the immunologic destruction of allografts remains a significant problem. We have recently identified a novel type of cytolytic T lymphocyte (CTL) that recognizes MHC class I alloantigens in a kidney-restricted manner; i.e., these alloreactive CTL lyse kidney cell targets at high levels but show little reactivity to lymphoid cell targets. Previous studies of CTL activity have utilized lymphoid targets almost exclusively; consequently, the fundamental characteristics of parenchymal cell-restricted effector populations and their role in allograft rejection are unknown. Our ultimate goal is to determine the role of parenchymal cell-restricted effector populations in the clinical rejection of organ transplants. To facilitate the investigation of these issues, we have established a mouse model of kidney-restricted allorecognition. We have already isolated and characterized mouse CTL-KR clones, and have now established that CTL-KR dominate the murine CTL response to allogeneic kidney cells in vitro. The current proposal will utilize rodent models to elucidate five key aspects of CTL-KR immunobiology. Highly-focused clinical studies can then follow to verify relevance to the human system. To determine if CTL-KR can mediate tissue-detruction in vivo, mouse CTL-KR clones will be tested for the capacity to destroy functional elements of the kidney following injection under the kidney capsule. Rat kidney transplant models that mimic acute, delayed, and chronic allograft rejection will be used to define the in vivo conditions that elicit CTL-KR; the spontaneous kidney- restricted lytic activity of graft infiltrating cells will be examined in these experiments. Human CTL-KR clones will be propagated from clinical rejection biopsies to verify that they have the same properties as those from the mouse and normal humans. Limiting dilution analyses will be used to quantitate the contribution of CTL-KR to the total alloreactive CTL repertoire, and to determine if CTL-KR are elicited by conventional APC. To determine which cell types present within a graft elicit tissue- restricted CTL, purified populations of parenchymal cells (hepatocytes, keritinocytes, pancreatic islets) and leukocytes (macrophages, B-cells, and dendritic cells) will be tested for the capacity to elicit kidney- restricted CTL responses; CTL generation assays will be used to define the costimulatory requirements for activation of CTL-KR from purified CD8+ T- cell precursors. Monoclonal antibodies to T-cell differentiation antigens will be used in FACS analysis and CTL blocking studies to determine if CTL- KR utilize tissue-restricted accessory molecule/ligand interactions for target cell lysis; mHC-associated peptides will be isolated from kidney cells and tested for the capacity to sensitize lymphoid cells to lysis by CTL-KR. Together, these experiments will provide important new information on the cellular basis of clinical graft loss.